Anti fosb

The following were purchased: PIPES, bovine serum albumin (BSA), EGTA, EDTA, D-glucose, NaF, Na₃VO₄, 2-ME (2-mercaptoethanol); RPMI 1640 containing 25 mM HEPES and L-glutamine (BioWhittaker, Walkersville, MD); Percoll (Pharmacia, Piscataway, NJ); Tris(hydroxymethyl)-aminomethane, Tween-20 (Bio-Rad,Hercules, CA); anti-CD25 alpha subunit and anti-CD25 beta subunit (CD122) (AbD Serotec, Raleigh, NC); anti-TRPM2 (Genetex, Irvine, Ca); anti-maxi-K channel, (Abcam, Cambridge, UK); anti-FosB (Cell Signaling, Beverly, MA), anti-Egr-3 (Santa Cruz Antibodies, Santa Cruz, CA), anti-HSP90beta (Millipore, Temcula, CA), anti-Orai1 and anti-STIM1 (Proteintech Group, Inc. Chicago, Il.), HRP-conjugated sheep anti-mouse Ig Ab (Amersham Life Science, Arlington Heights, IL). Mouse anti-human IgE Ab (6061P) (Hybridoma Farms, MD). PIPES-albumin-glucose (PAG) buffer consisted of 25 mM PIPES, 110 mM NaCl, 5 mM KCl, 0.1% glucose, and 0.003% HSA. PAGCM was PAG supplemented with 1 mM CaCl₂ and 1 mM MgCl₂. Countercurrent elutriation and labeling with antibodies for flow cytometry was conducted in PAG containing 0.25% BSA in place of 0.003% HSA (elutriation buffer). ESB is Novex electrophoresis sample buffer containing 5% 2-mercaptoethanol. Buffers were prepared with RNase and DNase-free reagents where possible.

The esophageal squamous cell carcinoma (ESCC) cells KYSE140, KYSE520, and TE1 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were subjected to mycoplasma examination and cytogenetically test and cultured with completed culture medium (basic medium supplemented with 10% fetal bovine serum) according to the standard protocols. The compound isoliquiritigenin was purchased from Selleck Chemicals (Houston, TX). Recombinant human EGF was product of R&D. The primary antibodies used in this study including anti-p-EGFR (Tyr1068) (#3777), anti-EGFR (#4267), anti-p-Akt (S473) (#4060), anti-Akt (#4691), anti-p-ERK1/2 (Thr202/Tyr204) (#4370), anti-ERK1/2(#4695), anti-c-Jun (#9165), anti-JunB (#3746), anti-JunD (#5000), anti-FosB(#4691), anti-c-Fos (#2251), anti-Fra1(#5281), anti-Cyclin D1(#55506), anti-β-actin (#3700), and anti-Histone H3 Ser10 (#53348) were products of Cell Signaling Technology (Danvers, MA). Lentivirus plasmids (pLKO.1-shEGFR) were purchased from Thermo Scientific (Huntsville, AL, USA).